Skin Care Compositions and Methods Comprising Selective Agonists of Melanocortin 1 Receptor

ABSTRACT

Short tri- and tetrapeptides according to the following Formula I Ar(CH 2 ) m X 1 —X 2 —CO—X 3 —X 4 —X 5 -(Trp) n -NX 6 R are potent, selective agonists of melanocortin 1 receptor (MC1R). Provided herein are skin care compositions including Formula I peptide agonists of MC1R and methods of regulating a skin condition of a mammal that include applying to a treatment surface of the body a safe and effective amount of a skin care composition including a Formula I peptide. The peptides, skin care compositions, and skin care methods described herein are useful in regulating a skin condition of a mammal associated with exposure ultraviolet (UV) radiation, including sunburn, UV sensitivity, photoaging, and skin pigmentation, particularly in the absence of sun exposure.

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/729,018, filed Nov. 21, 2012, which application is herebyincorporated by reference in its entirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The presently disclosed subject matter and its respective embodimentswere made with U.S. Government support under Grant No. R01CA114095,awarded by the National Cancer Institute (NCI). The government hascertain rights in this invention.

FIELD OF THE INVENTION

The presently disclosed subject matter relates to the field ofmelanocortin 1 receptor (MC1R) agonists. Specifically, the presentinvention relates to skin care compositions comprising selective tri-and tetrapeptide agonists of MC1R useful in regulating a skin conditionresulting from exposure to UV radiation, such as sunburn, UVsensitivity, photoaging, and skin pigmentation, particularly stimulatingpigmentation without sun exposure (i.e., sunless tanning).

BACKGROUND OF THE INVENTION

The negative effects of long term exposure to ultraviolet (UV) radiationare well known. Nevertheless, many individuals desire the appearance oftanned skin. Other than traditional sunbathing, one popular method foracquiring a tan is through the use of tanning beds. However, thispractice has hazardous effects, since the utilized UVA rays are the mainculprit for photoaging and are promoters for skin cancer, particularlymelanoma. As a result, a need has developed for skin care compositionsthat provide a sunless tanning benefit, without the need for exposingthe skin to the sun's harmful rays.

Current sunless tanning options generally function by temporarily dyeingthe skin. However, various problems with current formulations remain.Because popular sunless tanning compositions are dyes, users must takecare not to stain clothing or towels. If not applied evenly, anundesirable streaked or blotchy appearance can result. Further, manyusers complain that the sunless tanning effect is short-lived and thatthe product must be frequently reapplied. Perhaps the most commoncomplaint is that the sunless tanning benefit appears artificial anddoes not resemble an authentic tan. Thus, the need for improved sunlesstanning options persists.

The melanocortin 1 receptor (MC1R) belongs to the family of melanocortinreceptors (subfamily of G-protein coupled receptors, or GPCRs) comprisedof five members (MC1R-MC5R), each encoded by a different gene. Thesereceptors vary widely in their expression and tissue distribution, withthe MC3R and MC4R being neuronal receptors, the MC2R predominantlyexpressed by the adrenal gland, and the MC1R and MC5R expressed in theskin. Alpha-melanocyte stimulating hormone (alphα-MSH) is a commonnative agonist for MC1R, MC3R, MC4R, and MC5R.

MC1R is expressed on the cell surface of melanocytes, cells that residein the upper layer of the skin. Melanocytes provide photoprotection bysynthesizing the pigment melanin that reduces the penetration of UVradiation and scavenges reactive oxygen radicals.

MC1R agonists work to protect the skin in two ways, enhancing repair ofDNA damage and stimulating production of melanin by melanocytes. Studieshave previously shown that activation of MC1R by its native,non-selective peptide ligand alpha melanocortin stimulating hormone(α-MSH) increases melanin synthesis. Treatment (6 h-8 h) of humanmelanocytes with α-MSH up-regulates expression of the MC1R gene, thusenhancing response of melanocytes to melanocortins. Further studies haveshown that α-MSH enhances repair of UV-induced DNA photoproducts, whichis expected to reduce mutations and malignant transformation, andinhibits apoptosis, and hence increases survival of human melanocyteswith undamaged DNA.

For compounds targeting the skin, selectivity for MC1R is of particularimportance. MC5R, while also expressed in the skin, is expressed on thesebaceous glands; its activation leads to increased sebum production,which causes acne. Zhang, L., et al., Melanocortin-5 receptor andsebogenesis, Eur. J. Pharmacol. 660(1): 202-06 (2011). Equally importantis selectivity for MC1R versus MC3R and MC4R; commercial development ofthe potent but non-selective synthetic α-MSH analog melanotan II (MT-II)for sunless tanning was restricted by its off-target effects thatincluded sexual arousal and spontaneous erections lasting for 1-5 hours,nausea, grade II somnolence, fatigue, stretching, yawning, and loss ofappetite caused by undesired activation of MC3R and MC4R, in addition tothe desired activation of MC1R. Dorr, R. T., et al., Evaluation ofmelanotan II, a superpotent cyclic melanotropic peptide in a pilotphase-1 clinical study, Life Sci. 58(20): 1777-84 (1996); King, S. H.,et al., Melanocortin receptors, melanotropic peptides and penileerections, Curr. Top. Med. Chem. 7(11): 1098-1106 (2007); Pfaus, J. G.,et al., Selective facilitation of sexual solicitation in the female ratby a melanocortin receptor agonist, Proc. Nat'l. Acad. Sci. USA 101(27):10201-04 (2004); Yang, Y., et al., Molecular basis for the interactionof [Nle4,D-Phe7]melanocyte stimulating hormone with the humanmelanocortin-1 receptor, J. Biol. Chem. 272(37): 2300-10 (1997).

Hence, a need exists for improved, selective agonists of MC1R thatregulate skin conditions and provide skin benefits, including sunlessskin tanning, while avoiding the problems associated with tanning bedsor traditional sunless tanning compositions and the adverse effects ofnon-selective MC1R agonists.

SUMMARY OF THE INVENTION

Provided herein are selective peptide agonists of melanocortin 1receptor (MC1R) according to the formula:

Ar(CH₂)_(m)X¹—X²—CO—X³—X⁴—X⁵-(Trp)_(n)-NX⁶R  (Formula I)

or a dermatologically acceptable salt, solvate, or enantiomer thereof,wherein:

Ar is selected from the group consisting of unsubstituted or substitutedphenyl and 5- or 6-membered heteroaryl;

m is 0, 1, 2, or 3;

X¹ is absent or X¹ is selected from the group consisting of O, NR′, S,Se, and CR′R″ wherein R′ is selected from the group consisting of H,linear or branched C1-C4 alkyl, OH, and linear or branched C1-C4 O-alkyland R″ is selected from the group consisting of H and linear or branchedC1-C4 alkyl; or wherein CR′R″ is a C3-C6 cycloalkyl;

X² is absent or X² is selected from the group consisting of1,2-phenylene, 1,3-phenylene, 1,4-phenylene, and CQ′Q″ wherein Q′ and Q″are each independently selected from the group consisting of H andlinear or branched C1-C4 alkyl;

X³ is selected from the group consisting of unsubstituted or substitutedL-histidine (His), 3-(2-pyridyl)-L-alanine (2-PAL),3-(3-pyridyl)-L-alanine (3-PAL), 3-(4-pyridyl)-L-alanine (4-PAL),3-(2-thienyl)-L-alanine (2-Thi), 3-(3-thienyl)-L-alanine (3-Thi),3-(2-furyl)-L-alanine (2-FurAla), 3-(3-furyl)-L-alanine (2-FurAla),L-homoserine (HoSer), O-methyl-L-homoserine (HoSer(Me)), andL-allylglycine;

X⁴ is selected from the group consisting of unsubstituted or substitutedD-phenylalanine (D-Phe), L-alpha-MePhe, 3-(2-thienyl)-D-alanine(D-2-Thi), 3-(3-thienyl)-D-alanine (D-3-Thi), 3-(2-furyl)-D-alanine(D-2-FurAla), and 3-(3-furyl)-D-alanine (D-3-FurAla);

X⁵ is selected from the group consisting of unsubstituted or substitutedL-arginine (Arg) and L-citrulline;

n is 0 or 1;

X⁶ is selected from the group consisting of H, linear or branched C1-C4alkyl, and C3-C4 cycloalkyl; and

R is selected from the group consisting of H, linear or branched C1-C12alkyl, linear or branched C1-C12 arylalkyl, and C3-C5 cycloalkyl;

or wherein X⁶ and R together form a C3-C5 cycloalkyl.

Also provided herein are skin care compositions comprising the selectivepeptide agonists of MC1R disclosed herein and methods of regulating askin condition comprising topically applying to a treatment surface ofthe body in need of such treatment a skin care composition as disclosedherein.

These and other objects, features, embodiments, and advantages willbecome apparent to those of ordinary skill in the art from a reading ofthe following detailed description and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows HPLC data for LK-513 (Ph(CH₂)₃CO-His-(D-4-Bip)-Arg-NHMe)after 4 months storage at room temperature. Results indicate the peptideis highly stable and substantially free of degradation.

FIG. 2 shows dose-dependent stimulation of cAMP in human melanocytes bytetrapeptides LK-184 and LK-467. Results indicate maximal cAMP forLK-467 was achieved at 0.01 nM, compared to 10 nM for LK-184, a leadtetrapeptide.

FIG. 3 shows dose-dependent stimulation of cAMP in human melanocytes bythe tripeptides LK-511, LK-513, and LK-514, in comparison to alphα-MSH(α-MSH). Results indicate the tripeptides LK-511, LK-513, and LK-514were about ten times less potent than α-MSH in increasing cAMPformation, with LK-514 being more potent than LK-511 or LK-513

FIG. 4 shows dose-dependent stimulation of the activity of tyrosinase,the rate limiting enzyme for melanin synthesis, by LK-184 and LK-467.Results indicate that tetrapeptides LK-467 and LK-184 had comparableeffects in tyrosinase activity.

FIG. 5 shows dose-dependent stimulation of tyrosinase activity byLK-511, LK-513, and LK-514, in comparison to α-MSH. Results indicatethat tripeptides LK-511, LK-513, and LK-514 resulted in significantstimulation of tyrosinase activity beginning at 1 nM.

FIG. 6 shows repair of UV-induced DNA damage via reduction of the levelsof cyclobutane pyrimidine dimers (CPD), the major form of DNAphotoproducts, 48 hours post UV exposure of human melanocytes, with orwithout peptide treatment. Results show that LK-467 was as effective asLK-184 in reducing CPD levels 48 hours after exposure. Both peptides hadthe same effect at 1 nM on CPD removal as 10 nM α-MSH.

FIG. 7 shows repair of UV-induced DNA damage via reduction of the levelsof CPD 48 hours post UV exposure of human melanocytes, with or withoutpeptide treatment. The tripeptides LK-511, LK-513, and LK-514 hadsimilar effect at 100 nM compared to 10 nM α-MSH.

FIG. 8 shows repair of UV-induced oxidative DNA damage via reduction ofthe levels of 8-oxodG, the major form of damage, measured in humanmelanocytes 24 hours post UV exposure, with or without peptidetreatment. Results show that LK-467 was as effective as LK-184 inreducing 8-oxodG levels 24 hours after UV exposure.

FIG. 9 shows repair of oxidative UV-induced DNA damage via reduction ofthe levels of 8-oxodG measured in human melanocytes 24 hours post UVexposure, with or without peptide treatment. When tested at 100 nM,tripeptides LK-511 and LK-513 had similar effect, while LK-514 hadgreater effect than 1 nM α-MSH in reducing the levels of 8-oxodG.

DETAILED DESCRIPTION OF THE INVENTION

The details of one or more embodiments of the presently-disclosedsubject matter are set forth in this document. Modifications toembodiments described in this document, and other embodiments, will beevident to those of ordinary skill in the art after a study of theinformation provided in this document.

While the following terms are believed to be well understood by one ofordinary skill in the art, definitions are set forth to facilitateexplanation of the presently-disclosed subject matter.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the presently-disclosed subject matter belongs.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as reaction conditions, and so forth usedin the specification and claims are to be understood as being modifiedin all instances by the term “about.” Accordingly, unless indicated tothe contrary, the numerical parameters set forth in this specificationand claims are approximations that can vary depending upon the desiredproperties sought to be obtained by the presently-disclosed subjectmatter.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage ismeant to encompass variations of in some embodiments ±20%, in someembodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, insome embodiments ±0.5%, and in some embodiments ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethod.

It should be understood that every maximum numerical limitation giventhroughout this specification includes every lower numerical limitation,as if such lower numerical limitations were expressly written herein.Every minimum numerical limitation given throughout this specificationwill include every higher numerical limitation, as if such highernumerical limitations were expressly written herein. Every numericalrange given throughout this specification will include every narrowernumerical range that falls within such broader numerical range, as ifsuch narrower numerical ranges were all expressly written herein.

As used herein, the term “agonist” refers to an agent, such as apeptide, that triggers an agonist biological response when exposed to areceptor. In certain embodiments, peptides disclosed herein are agonistsof the melanocortin 1 receptor (MC1R) and elicit an agonist response inMC1R that mimics the response of the endogenous MC1R ligand, alphamelanocortin stimulating hormone (α-MSH).

The stereochemistry of the standard amino acids is defined by twopossible mirror image isomers or enantiomers. A natural amino acidsoccur in the L-stereoisomer form, with its mirror image D-stereoisomerrarely found in nature. The L- and D-amino acid convention is defined bymatching amino acid structure to the structures of L-glyceraldehyde andD-glyceraldehyde. The asymmetric alpha-carbon of an amino acid is thenaligned with the asymmetric second carbon of glyceraldehyde. Chemicallysimilar groups in the structure are oriented similarly, namely, theamino acid alpha-carboxyl group (alpha-COO—) is aligned parallel to thealdehyde group (—CHO) of glyceraldehyde. The amino acid alpha-aminogroup (alpha-NH₃+) is aligned parallel to the hydroxyl group linked tothe middle carbon of glyceraldehyde. Finally, the variable amino acidR-group is aligned with the methanol group of glyceraldehyde. In thisconfiguration, the alpha-amino group of every L-amino acid is located onthe left side and spatially above the alpha-carbon, as in the —OH grouplinked to the second asymmetric carbon of L-glyceraldehyde.

As used here, the terms “halo,” “halide,” or “halogen” refer to fluoro,chloro, bromo, and iodo groups.

As used herein, the term “aryl” as a group or part of a group refers to:(i) an optionally substituted monocyclic or multicyclic aromaticcarbocyclic moiety of about 6 to about 14 carbon atoms, such as phenylor naphthyl; or (ii) an optionally substituted partially saturatedmulticyclic aromatic carbocyclic moiety in which an aryl and acycloalkyl or cycloalkenyl group are fused together to form a cyclicstructure, such as a tetrahydronaphthyl, indenyl or indanyl ring. Arylgroups may be substituted with one or more aryl group substituents whichmay be the same or different, where “aryl group substituent” includes,for example, acyl, acylamino, alkoxy, alkoxycarbonyl, alkylenedioxy,alkylsulphinyl, alkylsulphonyl, alkylthio, aroyl, aroylamino, aryl,arylalkyloxy, arylalkyloxycarbonyl, arylalkylthio, aryloxy,aryloxycarbonyl, arylsulphinyl, arylsulphonyl, arylthio, carboxy, cyano,halo, heteroaroyl, heteroaryl, heteroarylalkyloxy, heteroaroylamino,heteroaryloxy, hydroxy, nitro, haloalkyl, and the like.

As used herein, “heteroaryl” as a group or part of a group refers to anoptionally substituted aromatic monocyclic or multicyclic organic moietyof about 5 to about 10 ring members in which one or more of the ringmembers is/are element(s) other than carbon, for example nitrogen,oxygen or sulphur. Examples of suitable optionally substitutedheteroaryl groups include optionally substituted benzimidazolyl, furyl,imidazolyl, isoxazolyl, isoquinolinyl, isothiazolyl, oxadiazolyl,pyrazinyl, pyridazinyl, pyrazolyl, pyridyl, pyrimidinyl, pyrrolyl,quinazolinyl, quinolinyl, 1,3,4-thiadiazolyl, thiazolyl, thienyl andtriazolyl groups. Heteroaryl groups may be substituted with one or moreheteroaryl group substituents which may be the same or different, where“heteroaryl group substituent” includes, for example acyl, acylamino,alkoxycarbonyl, alkylenedioxy, aroyl, aroylamino, aryl,arylalkyloxycarbonyl, aryloxycarbonyl, carboxy, cyano, halo,heteroaroyl, heteroaryl, heteroaroylamino, hydroxy, nitro,trifluoromethyl, and the like.

As used herein, “alkyl” means, unless otherwise specified, as a group orpart of a group, an aliphatic hydrocarbon group which may be linear orbranched having about 1 to about 12 carbon atoms (C1-C12) in the chain.In certain embodiments, alkyl groups have from 1 to about 6 carbon atoms(C1-C6). In a more specific embodiment, alkyl refers to linear orbranched alkyl having about 1 to about 4 carbon atoms (C1-C4) in thechain. Exemplary alkyl groups include methyl, ethyl, n-propyl, i-propyl,n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, 3-pentyl, heptyl, octyl,nonyl, decyl, dodecyl, and the like.

As used herein, the term “cycloalkyl” refers to an optionallysubstituted non-aromatic monocyclic or multicyclic ring system of atleast 3 carbon atoms. Particular monocyclic cycloalkyl rings includeC3-7cycloalkyl such as cyclopropyl, cyclopentyl, cyclohexyl andcycloheptyl. Exemplary multicyclic cycloalkyl rings includeperhydronaphthyl, adamant-(1- or 2-)yl and norbornyl and spirocyclicgroups (e.g. spiro[4,4]non-2-yl). The cycloalkyl group may besubstituted by one or more (e.g. 1, 2, or 3) substituents chosen from,for example, alkyl, aryl, arylalkyl, halo, halo substituted alkyl,hydroxyalkyl, hydroxy, alkoxy, and the like. In a specific embodiment,cycloalkyl refers to C3-C5 cycloalkyl.

As used herein, the term “alkoxy” refers to an alkyl-O— group in whichthe alkyl group is as described herein. Exemplary alkoxy groups includemethoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, heptoxy, and the like.

As used herein, “arylalkyl” refers to an aryl-alkyl- group in which thearyl and alkyl moieties are as previously described. In certainembodiments, arylalkyl groups contain a C1-C12 alkyl moiety. Exemplaryarylalkyl groups include benzyl, 2-phenethyl, naphthlenemethyl, and thelike.

Unless otherwise constrained by the definition of the individualsubstituent, all the above substituents should be understood as beingall optionally substituted.

Unless otherwise constrained by the definition of the individualsubstituent, the term “substituted” refers to groups substituted withfrom 1 to about 5 substituents independently selected from the groupconsisting of hydrogen, halo, cyano, hydroxy, alkoxy, thiol, carboxy,carbonyl, sulfonyl, linear or branched alkyl, cycloalkyl,heterocycloalkyl, acyl, alkene, alkyne, aryl, heteroaryl, amino, and thelike, any of the above substituents optionally being furthersubstituted.

As used herein, the term “a safe and effective amount” refers to anamount of a peptide or skin care composition sufficient to significantlyinduce a positive skin benefit, including independently or incombinations the benefits disclosed herein, but low enough to avoidserious side effects, i.e., to provide a reasonable benefit to riskratio, within the scope of sound judgment of the skilled artisan.

As used herein, the term “dermatologically acceptable” means that thecompositions or components thereof so described are suitable for use incontact with skin without undue toxicity, incompatibility, instability,allergic response, and the like.

The peptide agonists of MC1R and skin care compositions of the presentinvention are useful for topical application and for regulating one ormore skin conditions. Regulation of a skin condition, especially a humanskin condition, is often required due to conditions that may be inducedor caused by factors internal and/or external to the body. For instance,“regulating a skin condition” includes prophylactically regulatingand/or therapeutically regulating a skin condition, particularly a skincondition associated with exposure to UV radiation, and may involve oneor more of the following: sunburn, UV sensitivity, photoaging(sun-induced aging that differs from chronological aging in thatphotoaging occurs less in individuals with darker skin complexion,compared to individuals with lighter skin complexion), discolorationsdue to melanin (e.g., pigment spots, age spots, uneven pigmentation,vitiligo), and skin pigmentation, particularly stimulating skinpigmentation without sun exposure (i.e., sunless tanning). As usedherein, prophylactically regulating skin condition includes delaying,minimizing and/or preventing harmful skin effects of exposure to UVradiation. As used herein, therapeutically regulating skin conditionincludes ameliorating, e.g., diminishing, minimizing and/or effacing,harmful skin effects of exposure to UV radiation. Regulating skincondition involves improving skin appearance and/or feel, for example,by providing a sunless tanning skin benefit.

MC1R Agonists

Small peptide agonists according to the following formula aresurprisingly potent and selective agonists of MC1R:

Ar(CH₂)_(m)X¹—X²—CO—X³—X⁴—X⁵-(Trp)_(n)-NX⁶R  (Formula I)

or a dermatologically-acceptable salt, solvate, or enantiomer thereof.

In certain embodiments, Ar is selected from phenyl or 5- or 6-memberedheteroaryl, any of which are optionally further substituted. In aspecific embodiment, Ar is phenyl or substituted phenyl. In anotherspecific embodiment, Ar is phenyl substituted with 1 to 5 substituents,each independently selected from the group consisting of halo, CN, OH,alkyl, cycloalkyl, haloalkyl, acyl, alkene, alkyne, alkoxy, aryl,heteroaryl, COY, COOY, SOY, SO₂Y′, and SO₂NYY′, wherein Y and Y′ areeach independently selected from H, alkyl, cycloalkyl, acyl, alkene, andalkyne, or wherein to X can optionally join to form a carbocyclic ringor a heterocyclic ring that is fused to Ar. In a very specificembodiment, Ar is selected from the group consisting of phenyl, andphenyl substituted with halo, alkyl, alkoxy, and OH.

In certain embodiments, m is 0, 1, 2, or 3. In a specific embodiment, mis 1 or 3, such that the peptide of Formula I contains a CH₂ or (CH₂)₃moiety.

In another embodiment, X¹ is absent or X¹ is selected from the groupconsisting of O, NR′, S, Se, and CR′R″ wherein R′ is selected from thegroup consisting of H, linear or branched C1-C4 alkyl, OH, and linear orbranched C1-C4 O-alkyl and R″ is selected from the group consisting of Hand linear or branched C1-C4 alkyl; or wherein CR′R″ is a C3-C6cycloalkyl.

In another embodiment, X² is absent or X² is selected from the groupconsisting of 1,2-phenylene, 1,3-phenylene, 1,4-phenylene, and CQ′Q″wherein Q′ and Q″ are each independently selected from the groupconsisting of H and linear or branched C1-C4 alkyl.

In certain embodiments, X³ is selected from the group consisting ofunsubstituted or substituted L-histidine (His), 3-(2-pyridyl)-L-alanine(2-PAL), 3-(3-pyridyl)-L-alanine (3-PAL), 3-(4-pyridyl)-L-alanine(4-PAL), 3-(2-thienyl)-L-alanine (2-Thi), 3-(3-thienyl)-L-alanine(3-Thi), 3-(2-furyl)-L-alanine (2-FurAla), 3-(3-furyl)-L-alanine(2-FurAla), L-homoserine (HoSer), O-methyl-L-homoserine (HoSer(Me)), andL-allylglycine. In a specific embodiment, X³ is L-Histidine (His).

In certain embodiments, X⁴ is selected from the group consisting ofunsubstituted or substituted D-phenylalanine (D-Phe) or a bulkyhydrophobic analog of D-Phe. While not desiring to be bound by theory,it is believed that bulky aromatic rings of certain substituted D-Phemoieties (e.g., D-4-tBuPhe, D-4-Bip, D-1-Nal, D-2-Nal, and the like)provide favorable interaction with a hydrophobic pocket of MC1R andprevent peptides of Formula I from binding with MC3R, MC4R, or MC5R.Further, the bulk and hydrophobicity of the X⁴ moiety shield the polaramid bonds of peptides of Formula I from chemical and enzymaticdegradation. Moreover, when X⁴ comprises an amino acid in the Dconfiguration, tripeptides of Formula I have no amide bonds with naturalamino acids, and tetrapeptides of Formula I have only one amide bondbetween natural amino acids (X⁵-Trp). While not desiring to be bound bytheory, it is believed that the absence or reduction of amide bondsbetween natural amino acids further protect peptides of Formula I fromchemical and enzymatic hydrolysis.

Accordingly, in certain embodiments, X⁴ is selected the group consistingof unsubstituted or substituted D-Phe, L-alpha-MePhe,3-(2-thienyl)-D-alanine (D-2-Thi), 3-(3-thienyl)-D-alanine (D-3-Thi),3-(2-furyl)-D-alanine (D-2-FurAla), and 3-(3-furyl)-D-alanine(D-3-FurAla). In certain embodiments, substituted D-Phe comprisesD-4-t-Bu-phenylalanine (D-4-tBuPhe), D-alpha-methylphenylalanine(D-alpha-MePhe), D-4-biphenylalanine (D-4-Bip), D-1-naphthylalanine(D-1-Nal), D-2-naphthylalanine (D-2-Nal), 4-FPhe, 4-ClPhe, 4-BrPhe,4-IPhe, 4-NO₂Phe, or 3-NO₂Phe. In a specific embodiment, X⁴ is selectedfrom the group consisting of D-Phe, D-2-Thi, D-3-Thi, D-4-tBuPhe,D-alpha-MePhe, L-alpha-MePhe, D-4-Bip, D-1-Nal, and D-2-Nal. In a morespecific embodiment, X⁴ is selected from the group consisting of D-Phe,D-4-tBuPhe, D-4-Bip, D-1-Nal, and D-2-Nal. In a more specificembodiment, X⁴ is selected from the group consisting of D-4-tBuPhe,D-4-Bip, D-1-Nal, and D-2-Nal. In certain embodiments, X⁴ is in the Dstereoisomer configuration.

In some embodiments, X⁵ is selected from the group consisting ofunsubstituted or substituted L-arginine (Arg) and L-citrulline. In aspecific embodiment, X⁵ is L-Arginine (Arg).

Formula I peptides comprise both tri- and tetrapeptides. In certainembodiments, the peptides of Formula I are tripeptides, wherein Trp isabsent and n is 0. In other embodiments, the peptides of Formula I aretetrapeptides, wherein Trp is present and n is 1.

In certain embodiments, X⁶ is selected from the group consisting of H,linear or branched C1-C4 alkyl, and C3-C4 cycloalkyl. In a specificembodiment, X⁶ is selected from the group consisting of H and linear orbranched C1-C3 alkyl. In a more specific embodiment, X⁶ is selected fromthe group consisting of H, methyl, and ethyl.

In another embodiment, R is selected from the group consisting of H,linear or branched C1-C12 alkyl, linear or branched C1-C12 arylalkyl,and C3-C5 cycloalkyl. In a specific embodiment, R is selected from thegroup consisting of H and linear or branched C1-C3 alkyl. In a morespecific embodiment, R is selected from the group consisting of H,methyl, and ethyl.

Alternatively, in some embodiments X⁶ and R taken together form a C3-C5cycloalkyl.

In another embodiment, MC1R agonist peptides are provided according tothe following formula:

Ph(CH₂)₃CO-(His)-(X⁴)-(Arg)-(Trp)_(n)-NHR  (Formula II)

or a dermatologically acceptable salt, solvate, or enantiomer thereof,wherein:

Ph is unsubstituted or substituted phenyl;

X⁴ is selected from the group consisting of D-phenylalanine (D-Phe),D-1-naphthylalanine (D-1-Nal), D-4-biphenylalanine (D-4-Bip), andD-4-t-butylphenylalanine (D-4-tBuPhe);

n is 0 or 1; and

R is selected from the group consisting of H, methyl, and ethyl.

In certain embodiments, R is methyl or ethyl when X⁴ is D-Phe.

In a specific embodiment, MC1R agonists are selected from the peptidesprovided in Table 2, below. In a very specific embodiment, MC1R agonistsare selected from the group consisting ofPh(CH₂)₃CO-His-(D-Phe)-Arg-Trp-NHEt (LK-487),Ph(CH₂)₃CO-His-(D-1-Nal)-Arg-Trp-NH₂ (LK-467),Ph(CH₂)₃CO-His-(D-4-Bip)-Arg-NH₂ (LK-511),Ph(CH₂)₃CO-His-(D-4-Bip)-Arg-NHMe (LK-513), andPh(CH₂)₃CO-His-(D-4-tBuPhe)-Arg-NH₂ (LK-514).

In another embodiment, the peptides of Formula I and Formula II aretripeptides that lack tryptophan (Trp), such that n=0. Surprisingly, ithas been found that short Formula I and Formula II tripeptides lackingTrp are particularly selective for MC1R over other melanocortinreceptors. Without being bound by theory, it is believed that thepresence of Trp may be a factor in agonism of MC3R, MC4R, and MC5R, butmay not be as critical for agonism of MC1R. Hence, removal of the Trpmoiety may yield short tripeptide agonists of MC1R that avoid agonism ofMC3R, MC4R, and MC5R, which has significant clinical implications.

Synthesis

Formula I and Formula II peptides disclosed herein are synthesized usingsolid-phase synthesis and Fmoc chemistry on Rink amide resin forunsubstituted amide peptides and alkyl indole resin for alkyl amidepeptides (R=Me or Et) in dichloromethane, dimethylformamide (DMF) orN-methylpiperidone (NMP). The Fmoc groups are removed by piperidinesolutions, benzotriazol-1-yloxy)tris(dimethylamino)phosphoniumhexafluorophosphate or tetrafluoroborate (BOP) orO-benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluorophosphate (HBTU)are used as coupling agents and N-methylmorpholine (NMM) orN,N-diisopropylethylamine (DIPEA) as bases with optional addition ofN-hydroxybenzotriazole (NHOBT). The crude peptides are cleaved from theresin with simultaneous deprotection with 95% trifluoroacetic acid(TFA)+triisopropyl silane (TIS) or 2-mercaptoethanol. Preparative HPLC(detection at 254 nm) is performed on 250×50 mm 10 μm Polaris C18-Acolumn with 40 min gradient from 6 to 60% MeCN in water with 0.1% TFA at60 mL/min. Analytical HPLC (detection at 214 and 254 nm) is performed at1 mL/min on Altex 5 μm C18 column, 4.6×250 mm, 13 min gradient from 5 to95% MeCN in water with 0.1% H₃PO₄. Identity of synthesized peptides isconfirmed by mass spectrometry (MS).

Tri- and Tetrapeptide Secondary Structure

Natural agonists of melanocortin receptors, including alpha-, beta-, andgamma-melanocyte stimulating hormones, share the common melanocortincore sequence His⁶-Phe⁷-Arg⁸-Trp⁹ (HFRW), existing in a beta-turnconformation. Introduction of D-Phe⁷ (HfRW) into the core sequence isbelieved to stabilize the beta-turn conformation in certain syntheticMSH analogs (NDP-MSH, MT-II), which increases potency. However, despiteincreased potency, the natural ligands and those analogs lackselectivity for MC1R.

Surprisingly, it has been found that rigidifying the peptide backbone oftri- and tetrapeptides disclosed herein by introducing beta-turninducing amino acids leads to decreased activity of the peptides.Satyanarayanajois S. D., et al., Conformations of end-cappedmelanocortin agonists RCO-X-Z-Arg-Trp-NH ₂ by 2D-NMR, CD andcomputations, Peptides Breaking Away: Proceedings of the Twenty-FirstAmerican Peptide Association 384 (2009). Further, “soft fixation” of thetripeptide trans-4-HOC₆H₄CH═CHCO-His-D-Phe-Arg-NH₂ conformation withoutrestricting dihedral angles of the backbone or side chains resulted in acompound with moderately increased MC1R selectivity and potency at mouseMCRs compared to the tripeptide LK-394. Ruwe A. R. et al., Semi-rigidtripeptide agonists of melanocortin receptors, Bioorg. Med. Chem. Lett.19(17): 5176-81 (2009). However, this peptide proved to be less potentin human melanocytes. Thus, while not desiring to be bound by theory, itis believed that the conformational restraints favorable for longerpeptides may be unfavorable for shorter MC1R agonists, including theFormula I and Formula II peptides disclosed herein.

Thus, in certain embodiments, it is desirable to select tri- andtetrapeptide agonists according to the formulas set forth herein thatare substantially free from conformational restraints imposed bysecondary peptide structure. That is, in certain embodiments, peptideagonists having a conformationally flexible structure (i.e., notconstrained by a beta-turn or any rigid conformation) show increasedpotency, binding activity, and selectivity for MC1R.

Skin Care Compositions

Provided herein are skin care compositions comprising (a) a safe andeffective amount of a Formula I or Formula II peptide, or combinationsthereof, and (b) one or more dermatologically acceptable carriers.

The phrase “dermatologically acceptable carrier,” as used herein, meansthat the carrier is suitable for topical application to the skin, hasgood aesthetic properties, is compatible with the actives disclosedherein and any other components, and will not cause any safety ortoxicity concerns. A safe and effective amount of carrier is from about50% to about 99.99%, specifically from about 60% to about 99.9%, andmore specifically from about 70% to about 98% of the composition.

The carrier can be in a wide variety of types. For example, emulsioncarriers, including, but not limited to, oil-in-water, water-in-oil,water-in-oil-in-water, and oil-in-water-in-oil emulsions, are usefulherein. In these emulsion carriers, silicone can also be used as theoil, thus yielding silicone-in-water, water-in-silicone,water-in-silicone-in-water and oil-in-water-in-silicone emulsions. Thesecarriers can also take many forms, non-limiting examples of whichinclude liquids, milks, serums, lotions, creams, sprays, aerosols,mousses, foams, sticks, gels, and pencils.

Emulsions according to the present invention generally contain anaqueous solution and a lipid or oil. Lipids and oils may be derived fromanimals, plants, or petroleum and may be natural or synthetic (i.e.,man-made, such as silicones). Specific emulsions also contain ahumectant, such as glycerin. Emulsions will optionally further containfrom about 0.05% to about 10%, more specifically from about 0.1% toabout 5%, of an emulsifier, based on the weight of the composition.Emulsifiers may be nonionic, anionic or cationic. Suitable emulsifiersare disclosed in, for example, McCutcheon's 2013 Emulsifiers andDetergents, North American Edition (2013). Suitable emulsions may have awide range of viscosities, depending on the desired product form.

In one embodiment, the concentration of Formula I or Formula II peptidesin a skin care composition ranges from about 1 mg/ml to about 1000 mg/mland includes all values and increments therebetween, especiallyincluding 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000mg/ml. In another embodiment, the concentration of Formula I or FormulaII peptides ranges from about 1 mg/ml to about 100 mg/ml, including allvalues and increments therebetween. In a specific embodiment, theconcentration of Formula II or Formula II peptides ranges from about 1mg/ml to about 50 mg/ml, including all values and incrementstherebetween. In a very specific embodiment, the concentration ofFormula II or Formula II peptides is about 40 mg/ml.

In certain embodiments, the pharmaceutical composition is a topicalcomposition comprising as a vehicle from about 20% to about 50%propylene glycol and from about 50% to about 80% water, with pH adjustedto 4.0 with 10 mM citric acid.

Methods of Use

The Formula I and Formula II MC1R peptide agonists and skin carecompositions comprising the same are useful in methods of regulating askin condition in a mammal.

In some embodiments, the method for regulating a skin conditioncomprises the step of topically applying to a treatment surface of thebody in need of such treatment a safe and effective amount of a FormulaI or Formula II MC1R peptide agonist disclosed herein, or combinationsthereof. The peptide agonist, as set forth above, includes the agonistand its dermatologically acceptable salts, solvates, and enantiomers. Insome embodiments, the peptide agonist or agonists are present in a skincare composition as described hereinabove.

The presently disclosed MC1R agonists are effective in regulating a widevariety of skin conditions, including but not limited to, sunburn, UVsensitivity, photoaging, and skin pigmentation. In a specificembodiment, regulating a skin condition comprises stimulating skinpigmentation in the absence of UV exposure (i.e., sunless tanning).

In some embodiments, a safe and effective amount of the peptide agonistsdescribed herein is delivered topically to the skin of an individual.When administered topically, the peptide agonists disclosed herein aresmall in size and lipophilic and are able to effectively penetrate theupper layers of the skin and contact the melanocytes located in thebasal layer of the epidermis.

In one embodiment, the peptides and compositions disclosed hereinregulate skin condition by stimulating production of melanin bymelanocytes, even in the absence of sun exposure, thereby providingincreased skin protection from ultraviolet radiation. Surprisingly, ithas been discovered that the peptide agonists disclosed herein alsodirectly activate DNA repair pathways, independently of the productionof melanin, as repair of DNA damage can be measured prior to increase inmelanin content of melanocytes. Also, these peptides prevent oxidativestress and oxidative DNA damage, thus inhibiting melanocyte death, as inthe case of vitiligo. Hence, the peptide agonists disclosed herein havewide application in regulating skin conditions associated with DNAdamage from UV radiation.

In addition, it will be appreciated that therapeutic benefits forregulating skin condition can be realized by combining treatment withone or more additional skin care active agents. In certain embodiments,the additional skin care active agent is selected from the groupconsisting of desquamatory actives, anti-acne actives, wrinkle repairactives, anti-oxidants, radical scavengers, chelators, anti-inflammatoryagents, topical anesthetics, anti-cellulite agents, flavonoids,antimicrobial actives, antifungal actives, sunscreen actives,conditioning agents, and combinations thereof.

In a specific embodiment, the additional skin care active agentcomprises a sunscreen active. As used herein, the term “sunscreenactive” refers to an agent applied to the skin to prevent sunburn bychemically blocking UV radiation from the sun. Many sunscreens are knownin the art and include, but are not limited to, titanium dioxide, zincoxide, PABA esters (glyceryl, padimate A and padimate O), salicylates(homosalate, octyl salicylate), cinnamates (cinoxate, octylmethoxycinnamate, octocrylene), benzophenones, ecamsule (Mexoryl™), andthe like.

Combination methods involving the peptides disclosed herein and anotherskin care active agent can be achieved by administering both agents atsubstantially the same time. Alternatively, combination methods with thepeptide agonists disclosed herein can precede or follow application ofthe other agent by intervals ranging from minutes to weeks.

Accordingly, in one embodiment, a selective peptide agonist ofmelanocortin 1 receptor (MC1R) according to the formula is provided:

Ar(CH₂)_(m)X¹—X²—CO—X³—X⁴—X⁵-(Trp)_(n)-NX⁶R  (Formula I)

or a dermatologically acceptable salt, solvate, or enantiomer thereof,wherein:

Ar is selected from the group consisting of unsubstituted or substitutedphenyl and 5- or 6-membered heteroaryl;

m is 0, 1, 2, or 3;

X¹ is absent or X¹ is selected from the group consisting of O, NR′, S,Se, and CR′R″ wherein R′ is selected from the group consisting of H,linear or branched C1-C4 alkyl, OH, and linear or branched C1-C4 O-alkyland R″ is selected from the group consisting of H and linear or branchedC1-C4 alkyl; or wherein CR′R″ is a C3-C6 cycloalkyl;

X² is absent or X² is selected from the group consisting of1,2-phenylene, 1,3-phenylene, 1,4-phenylene, and CQ′Q″ wherein Q′ and Q″are each independently selected from the group consisting of H andlinear or branched C1-C4 alkyl;

X³ is selected from the group consisting of unsubstituted or substitutedL-histidine (His), 3-(2-pyridyl)-L-alanine (2-PAL),3-(3-pyridyl)-L-alanine (3-PAL), 3-(4-pyridyl)-L-alanine (4-PAL),3-(2-thienyl)-L-alanine (2-Thi), 3-(3-thienyl)-L-alanine (3-Thi),3-(2-furyl)-L-alanine (2-FurAla), 3-(3-furyl)-L-alanine (2-FurAla),L-homoserine (HoSer), O-methyl-L-homoserine (HoSer(Me)), andL-allylglycine;

X⁴ is selected from the group consisting of unsubstituted or substitutedD-phenylalanine (D-Phe), L-alpha-MePhe, 3-(2-thienyl)-D-alanine(D-2-Thi), 3-(3-thienyl)-D-alanine (D-3-Thi), 3-(2-furyl)-D-alanine(D-2-FurAla), and 3-(3-furyl)-D-alanine (D-3-FurAla);

X⁵ is selected from the group consisting of unsubstituted or substitutedL-arginine (Arg) and L-citrulline;

n is 0 or 1;

X⁶ is selected from the group consisting of H, linear or branched C1-C4alkyl, and C3-C4 cycloalkyl; and

R is selected from the group consisting of H, linear or branched C1-C12alkyl, linear or branched C1-C12 arylalkyl, and C3-C5 cycloalkyl;

or wherein X⁶ and R together form a C3-C5 cycloalkyl.

In one embodiment, substituted D-Phe comprises D-4-t-Bu-phenylalanine(D-4-tBuPhe), D-alpha-methylphenylalanine (D-alpha-MePhe),D-4-biphenylalanine (D-4-Bip), D-1-naphthylalanine (D-1-Nal),D-2-naphthylalanine (D-2-Nal), 4-FPhe, 4-ClPhe, 4-BrPhe, 4-IPhe,4-NO₂Phe, or 3-NO₂Phe.

In another embodiment, Ar is selected from the group consisting ofunsubstituted or substituted phenyl and 5- or 6-membered heteroaryl; mis 1, 2, or 3; X¹ is absent; X² is absent; X³ is His; X⁴ is selectedfrom the group consisting of D-Phe, D-2-Thi, D-3-Thi, D-4-tBuPhe,D-alpha-MePhe, L-alpha-MePhe, D-4-Bip, D-1-Nal, and D-2-Nal; X⁵ isselected from the group consisting of unsubstituted or substituted Argand L-citrulline; n is 0 or 1; X⁶ is selected from the group consistingof H, linear or branched C1-C4 alkyl, and C3-C4 cycloalkyl; and R isselected from the group consisting of H, linear or branched C1-C12alkyl, linear or branched C1-C12 arylalkyl, and C3-C5 cycloalkyl.

In a specific embodiment, Ar is unsubstituted or substituted phenyl; mis 3; X¹ is absent; X² is absent; X³ is His; X⁴ is selected from thegroup consisting of D-4-tBuPhe, D-4-Bip, D-1-Nal, and D-2-Nal; X⁵ isunsubstituted or substituted Arg; n is 0 or 1; X⁶ is H, methyl, orethyl; and R is H, methyl, or ethyl.

In another embodiment, X⁴ is in the D stereoisomer configuration.

In still another embodiment, Ar is unsubstituted or substituted phenyl.In another embodiment, m is 3 and X¹ and X² are absent. In anotherembodiment, X³ is His. In another embodiment, X⁴ is selected from thegroup consisting of D-Phe, D-4-tBuPhe, D-4-Bip, D-1-Nal, and D-2-Nal. Inanother embodiment, X⁵ is Arg. In still another embodiment, X⁶ is H andR is H, methyl, or ethyl.

In a specific embodiment, the agonist is selected from the groupconsisting of Ph(CH₂)₃CO-His-(D-Phe)-Arg-Trp-NHEt;Ph(CH₂)₃CO-His-(D-1-Nal)-Arg-Trp-NH₂; Ph(CH₂)₃CO-His-(D-4-Bip)-Arg-NH₂;Ph(CH₂)₃CO-His-(D-4-Bip)-Arg-NHMe; andPh(CH₂)₃CO-His-(D-4-tBuPhe)-Arg-NH₂.

In another embodiment, the selective peptide agonist of MC1R issubstantially free from conformational restraints imposed by secondarystructure. In a specific embodiment, the selective peptide agonist ofMC1R is not constrained by a beta-turn conformation.

Also provided herein is a skin care composition for regulating skincondition of a mammal, comprising (a) a safe and effective amount of aFormula I or Formula II selective peptide agonist of MC1R, orcombinations thereof, as disclosed herein and (b) one or moredermatologically acceptable carriers. In a specific embodiment, the skincare composition is an emulsion. In a further embodiment, the skin carecomposition further comprises an additional skin care active selectedfrom the group consisting of desquamatory actives, anti-acne actives,wrinkle repair actives, anti-oxidants, radical scavengers, chelators,anti-inflammatory agents, topical anesthetics, anti-cellulite agents,flavonoids, antimicrobial actives, antifungal actives, sunscreenactives, conditioning agents, and combinations thereof.

Also provided herein is a method of regulating a skin condition of amammal, the method comprising the step of topically applying to atreatment surface of the body in need of such treatment a skin carecomposition as disclosed herein. In one embodiment, the skin conditionto be regulated is selected from the group consisting of sunburn, UVsensitivity, photoaging, and skin pigmentation. In a specificembodiment, regulating a skin condition comprises stimulating skinpigmentation in the absence of UV exposure (i.e., sunless tanning).

In a specific embodiment, the methods provided herein deliver theselective peptide agonist of MC1R to melanocytes in the skin. In oneaspect, the selective peptide agonist of MC1R stimulates production ofmelanin by melanocytes in the absence of sun exposure, thereby providingincreased skin protection from ultraviolet radiation. In another aspect,the selective peptide agonist of MC1R directly activates DNA repairpathways.

In another embodiment, the methods disclosed herein further compriseapplying a second skin care active agent. In a specific embodiment, thesecond skin care active agent is a sunscreen active.

EXAMPLES

The following examples are given by way of illustration and are in noway intended to limit the scope of the present invention.

Example 1 Selectivity of Peptide Agonists for Human Melanocortin 1Receptor (hMC1R)

Receptor selectivity of peptide agonists for hMC1R was determined bystimulation of cAMP in heterologous cells expressing different humanmelanocortin receptors (MC1R, MC3R, MC4R, and MC5R). Tested peptidesincluded the following: melanotan II (MT-II), alpha melanocortinstimulating hormone (α-MSH), LK-184(Ph(CH₂)₃CO-His-(D-Phe)-Arg-Trp-NH₂), LK-467(Ph(CH₂)₃CO-His-(D-1-Nal)-Arg-Trp-NH₂), LK-511(Ph(CH₂)₃CO-His-(D-4-Bip)-Arg-NH₂), LK-513(Ph(CH₂)₃CO-His-(D-4-Bip)-Arg-NHMe) and LK-514(Ph(CH₂)₃CO-His-(D-4-tBuPhe)-Arg-NH₂). Results are shown in Table 1below:

TABLE 1 Melanocortin Receptor Selectivity HMC1R HMC3R HMC4R HMC5R EC50 %MAX. EC50 % MAX. EC50 % MAX. EC50 % MAX. (NM) ACTIVITY (NM) ACTIVITY(NM) ACTIVITY (NM) ACTIVITY MT-II 3.57 103 — — — —  51.66 100  α-MSH — — 6.11 100  16.0 100 — — LK-184 0.19 128 22.7 85  1.69 102 262.3 39LK-467 0.34 111 32.4  7 18.1  39 122.0 34 LK-511 1.46 88 INACTIVE LK-5133.27 95 EC₅₀ > 10000 (2-7% MAX. ACTIVITY) LK-514 3.65 94

Results show that LK-467 is about ten times less potent in binding theundesirable hMC4R than tetrapeptide LK-184. Tripeptides LK-511, LK-513,and LK-514 are about as potent as MT-II with respect to activation ofMC1R, but are selective for activating MC1R and do not bind MC3R, MC4R,or MC5R. Results indicate that simultaneous introduction of hydrophobicsubstituents at the D-Phe position and the N- and/or C-terminus ofLK-184 results in more potent, selective MC1R peptide agonists.

Example 2 Peptide Stability

LK-513 (Ph(CH₂)₃CO-His-(D-4-Bip)-Arg-NHMe) was tested for stability anddegradation over a storage period. The peptide was stored in alyophilized form in open air at room temperature for 4 months. After 4months, HPLC was performed on a test sample of the peptide in order tomeasure degradation, using standard methods (FIG. 1). The peptide ishighly stable and resistant to degradation, showing only a single HPLCpeak. Without being bound by theory, it is believed that the enhancedstability is a product of the absence of amide bonds between naturalL-L-amino acids in the tripeptides and the existence of only one amidebond between natural amino acids in the tetrapeptides (Arg-Trp). Amidebonds between non-natural D- and natural L-amino acids are less prone tochemical and enzymatic hydrolysis, as evidenced by the FIG. 1. Further,the bulk and hydrophobicity of the X4 amino acid (D-Phe or a bulkyhydrophobic analog thereof, such as D-4-Bip in the case of LK-513)shield the polar amid bonds of the peptide from chemical and enzymaticdegradation.

Example 3 Dose-Dependent Stimulation of cAMP in Human Melanocytes

Peptides were tested on cultured human melanocytes for their capacity toactivate MC1R, as measured by stimulating cAMP formation. After 45minutes of treatment with different concentrations of peptides, cAMPlevels were measured using a I¹²⁵-labeled radioimmunoassay kit.Tetrapeptide LK-467 proved to be more potent than LK-184 in stimulatingcAMP formation, resulting in maximal levels of cAMP at a concentrationof 0.1 nM, compared to 10 nM of LK-184 (FIG. 2). Tetrapeptide LK-487 wasequipotent to LK-467 in this assay (data not shown). The tripeptidesLK-511, LK-513, and LK-514 were about ten times less potent than α-MSHin increasing cAMP formation, with LK-514 being more potent than LK-511or 513 (FIG. 3). These results demonstrate that these small peptideshave remarkable potency to activate the MC1R.

Example 4 Dose-Dependent Stimulation of Tyrosinase Activity

Peptides were tested on cultured human melanocytes for their capacityfor stimulating tyrosinase activity, the rate-limiting enzyme formelanin synthesis. Melanocytes were treated every other day, for a totalof 6 days, with increasing concentrations of peptides. Medium containingH³-tyrosine, the substrate for tyrosinase, was added to melanocytes 24hours before the end of the 6-day treatment. Tyrosinase activity wasmeasured by determining the amount of ³H₂O generated as H³-tyrosinase iscatalyzed by tyrosinase for melanin synthesis. Tetrapeptides LK-467 andLK-184 had comparable effects in tyrosinase activity, indicating thatLK-467 is at least 100 times more potent than α-MSH in activating MC1Rand stimulating melanogenesis (FIG. 4). Tetrapeptide LK-487 wasequipotent to LK-467 in this assay (data not shown). The tripeptidesLK-511, LK-513, and LK-514 resulted in significant stimulation oftyrosinase activity beginning at 1 nM, with LK-514 being the mosteffective (FIG. 5). These results demonstrate that these small peptideshave remarkable potency to activate MC1R and stimulate melanogenesis.

Example 5 Repair of UV-Induced Cyclobutane Pyrimidine Dimers

To investigate the effects of peptides on the repair of UV-induced DNAdamage, levels of cyclobutane pyrimidine dimers (CPD), the major form ofDNA photoproducts, were measured in UV-irradiated human melanocytes withor without peptide treatment. Melanocytes were pretreated with thepeptides for 4 days prior to, and for 48 hours post UV exposure,harvested, immunostained by a specific antibody for CPD, and analyzed byflow cytometry. Results show that LK-467 was as effective as LK-184 inreducing CPD levels 48 hours after exposure. Both peptides had the sameeffect at 1 nM on CPD removal as 10 nM α-MSH (FIG. 6). The tripeptidesLK-511, LK-513, and LK-514 at 100 nM had similar effect (FIG. 7). Theseresults demonstrate that these small peptides have profound effects onreducing levels of UV-induced DNA damage, thus reducing the chance ofmutations and malignant transformation of melanocytes to melanoma, amechanism expected to prevent melanoma formation.

Example 6 Repair of UV-Induced 8-Oxodeoxyguanosine (8-oxodG)

To investigate the effects of peptides on repair of UV-induced DNAdamage, levels of 8-oxodG, a major form of oxidative DNA damage, weremeasured in UV-irradiated human melanocytes with or without peptidetreatment. Melanocytes were pretreated with the respective peptide for 4days prior, and 24 days post UV exposure and immunostained with8-oxodG-specific antibody. Fluorescence intensity, which correlates withthe amount of DNA damage, was quantified using ImagJ software. Resultsshow that LK-467 was as effective as LK-184 in reducing 8-oxodG levels24 hours after exposure (FIG. 8). When tested at 100 nM, tripeptidesLK-511 and LK-513 had similar effect, while LK-514 had greater effectthan 1 nM α-MSH in reducing the levels of 8-oxodG (FIG. 9). Theseresults demonstrate that these small peptides have profound effects onreducing levels of UV-induced oxidative DNA damage, thus reducing thechance of mutations that lead to melanoma, and enhancing the survival ofmelanocytes to maintain photoprotection and prevent depigmentation, thehallmark of vitiligo.

Example 7 cAMP and Tyrosinase Activity

Peptides were tested as described in Example 4 above on cultured humanmelanocytes for their capacity to activate MC1R, as measured bystimulating cAMP formation, and for their capacity for stimulatingtyrosinase activity, the rate-limiting enzyme for melanin synthesis. Thetested peptides were synthesized according to the formula:

Ar(CH₂)_(m)X¹—X²—CO—X³—X⁴—X⁵-(Trp)_(n)-NX⁶R

wherein X¹ and X² are absent, X³ is His, X⁵ is Arg, and X⁶ is H. X⁴amino acids were in the D-stereoisomer form, unless otherwise noted.Results are shown in Table 2, below.

TABLE 2 cAMP and Tyrosinase Activity Compared to 10 nM a-MSH cAMP TAcompared compared to 10 nM to 10 nM LK Ar m X⁴ n R αMSH αMSH 480P4-FC6H4 1 Phe 1 H << < 205P 4-FC6H4 1 4-FPhe 1 H ≦ ≧ 252P 4-FC6H4 14-FPhe 1 Et NR 206P 4-HOC6H4 1 4-FPhe 1 H < 208P 4-BrC6H4 1 4-FPhe 1 H >210P 3,4-CH2O2 1 4-FPhe 1 H ≧ 470P Ph 3 4-FPhe 1 H > 473P Ph 3 4-ClPhe 1H =; > 474P Ph 3 4-BrPhe 1 H =; <; > 475P Ph 3 4-Iphe 1 H <; > 476P Ph 32-Thi 1 H > 467P Ph 3 1-Nal 1 H > 468P Ph 3 2-Nal 1 H ≦ 471P Ph 34-NO2Phe 1 H = 472P Ph 3 3-NO2Phe 1 H < 469P Ph 3 4-Bip 1 H ≦; > 480P4-FPh 1 Phe 1 H < 486P Ph 3 Phe 0 Et << 487P Ph 3 Phe 1 Et >* 488P 4-IPH1 Phe 1 H >> 489P 4-IPH 1 4-IPhe 0 H ≦ 491P Ph 3 1-Nal 0 Et NR 492P Ph 32-Nal 0 Et NR 493P Ph 3 4-Bip 0 Et << 494P Ph 3 4-tBuPhe 0 Et << 495P4-FPh 1 1-Nal 0 Et NR 496P 4-FPh 1 2-Nal 0 Et NR 497P 4-FPh 1 4-Bip 0 EtNR 498P 4-FPh 1 4-tBuPhe 0 Et NR** 503P 4-FPh 1 α-MePhe 0 Et < 510P Ph 31-Nal 0 H NR 511P Ph 3 4-Bip 0 H < < 512P Ph 3 1-Nal 0 Me NR 513P Ph 34-Bip 0 Me < < 514P Ph 3 4-tBuPhe 0 H < < 515P Ph 3 2-Nal 0 H << 516P Ph3 4-tBuPhe 0 Me << 517P Ph 3 2-Nal 0 Me <<< NR = No Response/inactive at10 nM *better responder than α-MSH **potential antagonist

Example 8 Tri- and Tetrapeptides

Examples of Formula I peptides are shown in Table 2, below.

TABLE 2 Formula I Peptides Ar(CH₂)_(m)X¹-X²-CO-X³-X⁴-X⁵-(Trp)_(n)-NX⁶RPh(CH₂)₃CO-His-(D-Phe)-Arg-Trp-NHEtPh(CH₂)₃CO-His-(D-1-Nal)-Arg-Trp-NHEtPh(CH₂)₃CO-His-(D-2-Nal)-Arg-Trp-NHEtPh(CH₂)₃CO-His-(D-4-Bip)-Arg-Trp-NHEtPh(CH₂)₃CO-His-(D-4-tBuPhe)-Arg-Trp-NHEt4-FC₆H₄CH₂CO-His-(D-Phe)-Arg-Trp-NHEt4-FC₆H₄CH₂CO-His-(D-1-Nal)-Arg-Trp-NHEt4-FC₆H₄CH₂CO-His-(D-2-Nal)-Arg-Trp-NHEt4-FC₆H₄CH₂CO-His-(D-4-Bip)-Arg-Trp-NHEt4-FC₆H₄CH₂CO-His-(D-4-tBuPhe)-Arg-Trp-NHEtPh(CH₂)₃CO-His-(D-Phe)-Arg-Trp-NHMe4-FC₆H₄CH₂CO-His-(D-Phe)-Arg-Trp-NHMePh(CH₂)₃CO-His-(D-1-Nal)-Arg-Trp-NH₂Ph(CH₂)₃CO-His-(D-2-Nal)-Arg-Trp-NH₂4-BrC₆H₄CH₂CO-His-(D-Phe)-Arg-Trp-NH₂4-FC₆H₄CH₂CO-His-(D-4-FPhe)-Arg-Trp-NH₂4-BrC₆H₄CH₂CO-His-(D-4-FPhe)-Arg-Trp-NH₂4-CF3C₆H₄CH₂CO-His-(D-4-FPhe)-Arg-Trp-NH₂3,4-O(CH₂)₂C₆H₄CH₂CO-His-(D-4-FPhe)-Arg-Trp-NH₂(D)-C₆H₄CH(OH)CO-His-(D-4-FPhe)-Arg-Trp-NH₂(L)-C₆H₄CH(OH)CO-His-(D-4-FPhe)-Arg-Trp-NH₂(D,L)-C₆H₄CH(OH)CO-His-(D-4-FPhe)-Arg-Trp-NH₂(D,L)-C₆H₄CH(OMe)CO-His-(D-4-FPhe)-Arg-Trp-NH₂(D)-4-ClC₆H₄CH(OH)CO-His-(D-4-FPhe)-Arg-Trp-NH₂(L)-4-ClC₆H₄CH(OH)CO-His-(D-4-FPhe)-Arg-Trp-NH₂(D,L)-4-ClC₆H₄CH(OH)CO-His-(D-4-FPhe)-Arg-Trp-NH₂C₆F₅CH₂CO-His-(D-4-FPhe)-Arg-Trp-NH₂4-C₆H₅C₆H₄CO-His-(D-4-FPhe)-Arg-Trp-NH₂4-C₆H₅OC₆H₄CO-His-(D-4-FPhe)-Arg-Trp-NH₂3-C₆H₅OC₆H₄CO-His-(D-4-FPhe)-Arg-Trp-NH₂Ph(CH₂)₃CO-His-(D-4-FPhe)-Arg-Trp-NH₂Ph(CH₂)₃CO-His-(D-4-ClPhe)-Arg-Trp-NH₂Ph(CH₂)₃CO-His-(D-4-BrPhe)-Arg-Trp-NH₂Ph(CH₂)₃CO-His-(D-4-IPhe)-Arg-Trp-NH₂Ph(CH₂)₃CO-His-(D-2-Thi)-Arg-Trp-NH₂Ph(CH₂)₃CO-His-(D-4-NO₂Phe)-Arg-Trp-NH₂Ph(CH₂)₃CO-His-(D-3-NO₂Phe)-Arg-Trp-NH₂4-IC₆H₄CH₂CO-His-(D-Phe)-Arg-Trp-NH₂4-IC₆H₄CH₂CO-His-(D-4-IPhe)-Arg-Trp-NH₂Ph(CH₂)₃CO-His-(D-Phe)-Arg-Trp-NH₂Ph(CH₂)₃CO-(4-PAL)-(D-Phe)-Arg-Trp-NH₂Ph(CH₂)₃CO-(2-Thi)-(D-Phe)-Arg-Trp-NH₂Ph(CH₂)₃CO-(3-Thi)-(D-Phe)-Arg-Trp-NH₂Ph(CH₂)₃CO-(2-FurAla)-(D-Phe)-Arg-Trp-NH₂Ph(CH₂)₃CO-(HoSer)-(D-Phe)-Arg-Trp-NH₂Ph(CH₂)₃CO-(HoSer(Me))-(D-Phe)-Arg-Trp-NH₂Ph(CH₂)₃CO-(AllGly)-(D-Phe)-Arg-Trp-NH₂ Ph(CH₂)₃CO-His-(D-1-Nal)-Arg-NH₂Ph(CH₂)₃CO-His-(D-2-Nal)-Arg-NH₂ Ph(CH₂)₃CO-His-(D-4-Bip)-Arg-NH₂Ph(CH₂)₃CO-His-(D-4-tBuPhe)-Arg-NH₂ 4-BrC₆H₄CH₂CO-His-(D-4-FPhe)-Arg-NH₂4-HOC₆H₄CH₂CO-His-(D-4-FPhe)-Arg-NH₂3,4-O(CH₂)₂C₆H₄CH₂CO-His-(D-4-FPhe)-Arg-NH₂Ph(CH₂)₃CO-His-(D-1-Nal)-Arg-NHMe Ph(CH₂)₃CO-His-(D-2-Nal)-Arg-NHMePh(CH₂)₃CO-His-(D-4-Bip)-Arg-NHMe Ph(CH₂)₃CO-His-(D-4-tBuPhe)-Arg-NHMePh(CH₂)₃CO-His-(D-1-Nal)-Arg-NHEt Ph(CH₂)₃CO-His-(D-2-Nal)-Arg-NHEtPh(CH₂)₃CO-His-(D-4-Bip)-Arg-NHEt Ph(CH₂)₃CO-His-(D-4-tBuPhe)-Arg-NHEt4-FC₆H₄CH₂CO-His-(D-Phe)-Arg-NHEt 4-FC₆H₄CH₂CO-His-(D-4-FPhe)-Arg-NHEt4-FC₆H₄CH₂CO-His-(D-1-Nal)-Arg-NHEt 4-FC₆H₄CH₂CO-His-(D-2-Nal)-Arg-NHEt4-FC₆H₄CH₂CO-His-(D-4-Bip)-Arg-NHEt4-FC₆H₄CH₂CO-His-(D-4-tBuPhe)-Arg-NHEt

Example 9 Formulation

An exemplary topical skin care composition is formulated as follows:

Ingredient Amount LK-511 40 mg/ml Propylene glycol 35% (v/v) Water 65%(v/v) 10 mM citric acid Adjust pH to 4.0

All documents cited are incorporated herein by reference; the citationof any document is not to be construed as an admission that it is priorart with respect to the present invention.

While particular embodiments of the present invention have beenillustrated and described, it would be obvious to one skilled in the artthat various other changes and modifications can be made withoutdeparting from the spirit and scope of the invention. It is thereforeintended to cover in the appended claims all such changes andmodifications that are within the scope of this invention.

1. A selective peptide agonist of melanocortin 1 receptor (MC1R)according to the following formula:Ar(CH₂)_(m)X¹—X²—CO—X³—X⁴—X⁵-(Trp)_(n)-NX⁶R  Formula I or adermatologically-acceptable salt, solvate, or enantiomer thereof,wherein: Ar is selected from the group consisting of unsubstituted orsubstituted phenyl and 5- or 6-membered heteroaryl; m is 0, 1, 2, or 3;X¹ is absent or X¹ is selected from the group consisting of O, NR′, S,Se, and CR′R″ wherein R′ is selected from the group consisting of H,linear or branched C1-C4 alkyl, OH, and linear or branched C1-C4 O-alkyland R″ is selected from the group consisting of H and linear or branchedC1-C4 alkyl; or wherein CR′R″ is a C3-C6 cycloalkyl; X² is absent or X²is selected from the group consisting of 1,2-phenylene, 1,3-phenylene,1,4-phenylene, and CQ′Q″ wherein Q′ and Q″ are each independentlyselected from the group consisting of H and linear or branched C1-C4alkyl; X³ is selected from the group consisting of unsubstituted orsubstituted L-histidine (His), 3-(2-pyridyl)-L-alanine (2-PAL),3-(3-pyridyl)-L-alanine (3-PAL), 3-(4-pyridyl)-L-alanine (4-PAL),3-(2-thienyl)-L-alanine (2-Thi), 3-(3-thienyl)-L-alanine (3-Thi),3-(2-furyl)-L-alanine (2-FurAla), 3-(3-furyl)-L-alanine (2-FurAla),L-homoserine (HoSer), O-methyl-L-homoserine (HoSer(Me)), andL-allylglycine; X⁴ is selected from the group consisting ofunsubstituted or substituted L alpha-MePhe, 3-(2-thienyl)-D-alanine(D-2-Thi), 3-(3-thienyl)-D-alanine (D-3-Thi), 3-(2-furyl)-D-alanine(D-2-FurAla), 3-(3-furyl)-D-alanine (D-3-FurAla), and substitutedD-phenylalanine (D-Phe); X⁵ is selected from the group consisting ofunsubstituted or substituted L-arginine (Arg) and L-citrulline; n is 0or 1; X⁶ is selected from the group consisting of H, linear or branchedC1-C4 alkyl, and C3-C4 cycloalkyl; and R is selected from the groupconsisting of H, linear or branched C1-C12 alkyl, linear or branchedC1-C12 arylalkyl, and C3-C5 cycloalkyl; or wherein X⁶ and R togetherform a C3-C5 cycloalkyl.
 2. The selective peptide agonist of MC1R ofclaim 1, wherein substituted D-Phe comprises D-4-t-Bu-phenylalanine(D-4-tBuPhe), D-alpha-methylphenylalanine (D-alpha-MePhe),D-4-biphenylalanine (D-4-Bip), D-1-naphthylalanine (D-1-Nal),D-2-naphthylalanine (D-2-Nal), 4-FPhe, 4-ClPhe, 4-BrPhe, 4-IPhe,4-NO₂Phe, or 3-NO₂Phe.
 3. The selective peptide agonist of MC1R of claim2, wherein: Ar is selected from the group consisting of unsubstituted orsubstituted phenyl and 5- or 6-membered heteroaryl; m is 1, 2, or 3; X¹is absent; X² is absent; X³ is His; X⁴ is selected from the groupconsisting of D-2-Thi, D-3-Thi, D-4-tBuPhe, D-alpha-MePhe,L-alpha-MePhe, D-4-Bip, D-1-Nal, and D-2-Nal; X⁵ is selected from thegroup consisting of unsubstituted or substituted Arg and L-citrulline; nis 0 or 1; X⁶ is selected from the group consisting of H, linear orbranched C1-C4 alkyl, and C3-C4 cycloalkyl; and R is selected from thegroup consisting of H, linear or branched C1-C12 alkyl, linear orbranched C1-C12 arylalkyl, and C3-C5 cycloalkyl.
 4. The selectivepeptide agonist of MC1R of claim 2, wherein: Ar is unsubstituted orsubstituted phenyl; m is 3; X¹ is absent; X² is absent; X³ is His; X⁴ isselected from the group consisting of D-4-tBuPhe, D-4-Bip, D-1-Nal, andD-2-Nal; X⁵ is unsubstituted or substituted Arg; n is 0 or 1; X⁶ is H,methyl, or ethyl; and R is H, methyl, or ethyl.
 5. (canceled)
 6. Theselective peptide agonist of MC1R of claim 1, wherein Ar isunsubstituted or substituted phenyl, m is 3, and X¹ and X² are absent.7. (canceled)
 8. The selective peptide agonist of MC1R of claim 1,wherein X³ is His and X⁵ is Arg.
 9. The selective peptide agonist ofMC1R of claim 1, wherein X⁴ is selected from the group consisting ofD-4-tBuPhe, D-4-Bip, D-1-Nal, and D-2-Nal.
 10. (canceled)
 11. Theselective peptide agonist of MC1R of claim 1, wherein X⁶ is H and R isH, methyl, or ethyl.
 12. A selective peptide agonist of melanocortin 1receptor (MC1R) selected from the group consisting of:Ph(CH₂)₃CO-His-(D-Phe)-Arg-Trp-NHEt;Ph(CH₂)₃CO-His-(D-1-Nal)-Arg-Trp-NH₂; Ph(CH₂)₃CO-His-(D-4-Bip)-Arg-NH₂;Ph(CH₂)₃CO-His-(D-4-Bip)-Arg-NHMe; andPh(CH₂)₃CO-His-(D-4-tBuPhe)-Arg-NH₂.
 13. The selective peptide agonistof MC1R of claim 1, wherein the agonist is substantially free fromconformational restraints imposed by secondary structure.
 14. Theselective peptide agonist of MC1R of claim 13, wherein the secondarystructure is not constrained by a beta-turn conformation.
 15. A skincare composition for regulating a skin condition of a mammal comprising:(a) a safe and effective amount of a selective peptide agonist of MC1Raccording to claim 1; and (b) one or more dermatologically acceptablecarriers.
 16. The skin care composition of claim 15, wherein the skincare composition is an emulsion.
 17. The skin care composition of claim15, further comprising an additional skin care active selected from thegroup consisting of desquamatory actives, anti-acne actives, wrinklerepair actives, antioxidants, radical scavengers, chelators,anti-inflammatory agents, topical anesthetics, anti-cellulite agents,flavonoids, antimicrobial actives, antifungal actives, sunscreenactives, conditioning agents, and combinations thereof
 18. A method ofregulating a skin condition of a mammal, the method comprising the stepof topically applying to a treatment surface of the body in need of suchtreatment the skin care composition according to claim
 15. 19. Themethod of claim 18, wherein the condition to be regulated is selectedfrom the group consisting of sunburn, ultraviolet (UV) sensitivity,photoaging, and skin pigmentation.
 20. The method of claim 19, whereinregulating a skin condition comprises stimulating skin pigmentation inthe absence of ultraviolet (UV) exposure.
 21. The method of claim 18,wherein the selective peptide agonist of MC1R stimulates production ofmelanin by melanocytes, thereby providing increased skin protection fromultraviolet radiation.
 22. The method of claim 18, wherein the selectivepeptide agonist of MC1R activates DNA repair pathways.
 23. A selectivepeptide agonist of melanocortin 1 receptor (MC1R) selected from thegroup consisting of: Ph(CH₂)₃CO-His-(D-Phe)-Arg-Trp-NHEt;Ph(CH₂)₃CO-His-(D-1-Nal)-Arg-Trp-NHEt;Ph(CH₂)₃CO-His-(D-2-Nal)-Arg-Trp-NHEt;Ph(CH₂)₃CO-His-(D-4-Bip)-Arg-Trp-NHEt;Ph(CH₂)₃CO-His-(D-4-tBuPhe)-Arg-Trp-NHEt;4-FC₆H₄CH₂CO-His-(D-Phe)-Arg-Trp-NHEt;4-FC₆H₄CH₂CO-His-(D-1-Nal)-Arg-Trp-NHEt;4-FC₆H₄CH₂CO-His-(D-2-Nal)-Arg-Trp-NHEt;4-FC₆H₄CH₂CO-His-(D-4-Bip)-Arg-Trp-NHEt;4-FC₆H₄CH₂CO-His-(D-4-tBuPhe)-Arg-Trp-NHEt;Ph(CH₂)₃CO-His-(D-Phe)-Arg-Trp-NHMe;4-FC₆H₄CH₂CO-His-(D-Phe)-Arg-Trp-NHMe;Ph(CH₂)₃CO-His-(D-1-Nal)-Arg-Trp-NH₂;Ph(CH₂)₃CO-His-(D-2-Nal)-Arg-Trp-NH₂;4-BrC₆H₄CH₂CO-His-(D-Phe)-Arg-Trp-NH₂;4-FC₆H₄CH₂CO-His-(D-4-FPhe)-Arg-Trp-NH₂;4-BrC₆H₄CH₂CO-His-(D-4-FPhe)-Arg-Trp-NH₂;4-CF3C₆H₄CH₂CO-His-(D-4-FPhe)-Arg-Trp-NH₂;3,4-O(CH₂)₂C₆H₄CH₂CO-His-(D-4-FPhe)-Arg-Trp-NH₂;(D)-C₆H₄CH(OH)CO-His-(D-4-FPhe)-Arg-Trp-NH₂;(L)-C₆H₄CH(OH)CO-His-(D-4-FPhe)-Arg-Trp-NH₂; (D,L)-C₆H₄CH(OH)CO-His-(D-4-FPhe)-Arg-Trp-NH₂; (D,L)-C₆H₄CH(OMe)CO-His-(D-4-FPhe)-Arg-Trp-NH₂;(D)-4-C1C₆H₄CH(OH)CO-His-(D-4-FPhe)-Arg-Trp-NH₂;(L)-4-C1C₆H₄CH(OH)CO-His-(D-4-FPhe)-Arg-Trp-NH₂; (D,L)-4-C1C₆H₄CH(OH)CO-His-(D-4-FPhe)-Arg-Trp-NH₂;C₆F₅CH₂CO-His-(D-4-FPhe)-Arg-Trp-NH₂;4-C₆H₅C₆H₄CO-His-(D-4-FPhe)-Arg-Trp-NH₂;4-C₆H₅OC₆H₄CO-His-(D-4-FPhe)-Arg-Trp-NH₂;3-C₆H₅OC₆H₄CO-His-(D-4-FPhe)-Arg-Trp-NH₂;Ph(CH₂)₃CO-His-(D-4-FPhe)-Arg-Trp-NH₂;Ph(CH₂)₃CO-His-(D-4-ClPhe)-Arg-Trp-NH₂;Ph(CH₂)₃CO-His-(D-4-BrPhe)-Arg-Trp-NH₂;Ph(CH₂)₃CO-His-(D-4-IPhe)-Arg-Trp-NH₂;Ph(CH₂)₃CO-His-(D-2-Thi)-Arg-Trp-NH₂;Ph(CH₂)₃CO-His-(D-4-NO₂Phe)-Arg-Trp-NH₂;Ph(CH₂)₃CO-His-(D-3-NO₂Phe)-Arg-Trp-NH₂;4-IC₆H₄CH₂CO-His-(D-Phe)-Arg-Trp-NH₂;4-IC₆H₄CH₂CO-His-(D-4-IPhe)-Arg-Trp-NH₂;Ph(CH₂)₃CO-(4-PAL)-(D-Phe)-Arg-Trp-NH₂;Ph(CH₂)₃CO-(2-Thi)-(D-Phe)-Arg-Trp-NH₂;Ph(CH₂)₃CO-(3-Thi)-(D-Phe)-Arg-Trp-NH₂;Ph(CH₂)₃CO-(2-FurAla)-(D-Phe)-Arg-Trp-NH₂;Ph(CH₂)₃CO-(HoSer)-(D-Phe)-Arg-Trp-NH₂;Ph(CH₂)₃CO-(HoSer(Me))-(D-Phe)-Arg-Trp-NH₂;Ph(CH₂)₃CO-(AllGly)-(D-Phe)-Arg-Trp-NH₂;Ph(CH₂)₃CO-His-(D-1-Nal)-Arg-NH₂; Ph(CH₂)₃CO-His-(D-2-Nal)-Arg-NH₂;Ph(CH₂)₃CO-His-(D-4-Bip)-Arg-NH₂; Ph(CH₂)₃CO-His-(D-4-tBuPhe)-Arg-NH₂;4-BrC₆H₄CH₂CO-His-(D-4-FPhe)-Arg-NH₂;4-HOC₆H₄CH₂CO-His-(D-4-FPhe)-Arg-NH₂;3,4-O(CH₂)₂C₆H₄CH₂CO-His-(D-4-FPhe)-Arg-NH₂;Ph(CH₂)₃CO-His-(D-1-Nal)-Arg-NHMe; Ph(CH₂)₃CO-His-(D-2-Nal)-Arg-NHMe;Ph(CH₂)₃CO-His-(D-4-Bip)-Arg-NHMe; Ph(CH₂)₃CO-His-(D-4-tBuPhe)-Arg-NHMe;Ph(CH₂)₃CO-His-(D-1-Nal)-Arg-NHEt; Ph(CH₂)₃CO-His-(D-2-Nal)-Arg-NHEt;Ph(CH₂)₃CO-His-(D-4-Bip)-Arg-NHEt; Ph(CH₂)₃CO-His-(D-4-tBuPhe)-Arg-NHEt;4-FC₆H₄CH₂CO-His-(D-Phe)-Arg-NHEt; 4-FC₆H₄CH₂CO-His-(D-4-FPhe)-Arg-NHEt;4-FC₆H₄CH₂CO-His-(D-1-Nal)-Arg-NHEt;4-FC₆H₄CH₂CO-His-(D-2-Nal)-Arg-NHEt;4-FC₆H₄CH₂CO-His-(D-4-Bip)-Arg-NHEt; and4-FC₆H₄CH₂CO-His-(D-4-tBuPhe)-Arg-NHEt.